PROJECT SUMMARY Fluorescent organic nanoparticles (FONPs) are a relatively new class of imaging probes that have unique potential for biomedical imaging applications because they integrate the synthetic flexibility of small organic molecules with the superior fluorescence properties of nanoparticles. FONPs have notable distinctions, however, compared to other types of fluorescent materials. Compared to the majority of organic dye fluorophores that display quenched fluorescence when aggregated, the fluorescent monomers that comprise FONPs have increased signal upon their self-assembly. Relative to other types of fluorescent NPs, such as quantum dots, FONPs have high quantum yield, can be optimized for size, color, and surface coating, and do not require the use of toxic elements. These factors taken together suggest that FONPs have great potential as a platform imaging technology. Indeed, FONPs have been used experimentally for tumor detection, apoptosis assessment, and cell tracking. Despite these early successes, significant innovations must be made to precisely control optical properties including wavelength and brightness, FONP self-assembly and overall size, and development of an easily adaptive surface coating for conjugation specific to the intended application. Thus, the overarching goal of this IMAT R21 proposal is to synthesize, characterize, and evaluate a new class of fluorescent organic nanoparticles (FONPs) as a novel imaging agents with controlled size, emission wavelengths, surface chemistry, and high quantum yield for diverse cancer imaging applications. Further, we hypothesize that FONPs with tunable fluorescence emission, size minimization, paramagnetic cores, and a clickable surface coating will provide targetable nanoparticle-based technology with bright and stable fluorescence emission for multiplexed and multimodality imaging. The goals, hypothesis, and innovation are addressed in two specific aims: (1) To design clickable, optically tuned, self-assembled fluorescent organic nanoparticles; (2) To validate the capacity of FONPs for multiplexed cellular imaging to differentiate inflammatory and tumor-associated neurolymphatic remodeling. Using neurolymphatic remodeling as the first application of the FONPs serves multiple purposes. It necessitates synthesizing distinct colors of FONPs for simultaneous imaging of more than one cell population, requires at least two types of ?clickable? targeting ligands, and allows for a quantifiable comparison with fluorescent protein-labeled cells and ligands conjugated to either small organic dyes or QDs. Finally, the 2D, 3D, and ex vivo results obtained with this proposal will provide additional insight into distinguishing inflammation from tumorigenesis using FONPs as a platform technology. If the quantitative milestones of this IMAT R21 are achieved, it will directly support their continued preclinical and clinical development.